Structural basis for the role of C-terminus acidic tail of Saccharomyces cerevisiae ubiquitin-conjugating enzyme (Rad6) in E3 ligase (Bre1) mediated recognition of histones

Yadav P, Gupta M, Wazahat R, Islam Z, Tsutakawa S, Kamthan M, Kumar P, International Journal of Biological Macromolecules :127717 (2023) DOI

SASDJA5 – E2 ubiquitin conjugating enzyme RAD6

Ubiquitin-conjugating enzyme E2 2
MWexperimental 20 kDa
MWexpected 20 kDa
VPorod 31 nm3
log I(s) 3.09×102 3.09×101 3.09×100 3.09×10-1
Ubiquitin-conjugating enzyme E2 2 small angle scattering data  s, nm-1
ln I(s)
Ubiquitin-conjugating enzyme E2 2 Guinier plot ln 3.10×102 Rg: 2.3 nm 0 (2.3 nm)-2 s2
(sRg)2I(s)/I(0)
Ubiquitin-conjugating enzyme E2 2 Kratky plot 1.104 0 √3 sRg
p(r)
Ubiquitin-conjugating enzyme E2 2 pair distance distribution function Rg: 2.2 nm 0 Dmax: 6.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Ubiquitin-conjugating enzyme E2 2 SREFLEX model
log I(s)
 s, nm-1
Ubiquitin-conjugating enzyme E2 2 SREFLEX model
log I(s)
 s, nm-1
Ubiquitin-conjugating enzyme E2 2 SREFLEX model
Synchrotron SAXS data from solutions of E2 ubiquitin conjugating enzyme RAD6 in 50 mM Tris, 150 mM NaCl, 1 mM TCEP, pH 7.5 were collected on the 12.3.1 (SIBYLS) beam line at the Advanced Light Source (ALS; Berkeley, CA, USA) using a MAR 165 CCD detector at a sample-detector distance of 1.6 m and at a wavelength of λ = 0.127 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 2.50 mg/ml was measured using exposure times of 1.0, 2.0, 3.0, 4.0 and 5.0 s at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering curve of buffer fraction was recorded as reference buffer scattering and was subtracted from sample scattering curve.

Protein was purified on Superdex 200 10/300 column in buffer condition 50 mM Tris pH 7.5 150 mM NaCl, 1mM TCEP. Each fraction was collected and concentrated with centrifugal concentration devices (10 kDa cut-off) to make the protein concentration up to 2.5 mg/ml. Fractions of buffer were also collected on Superdex 200 10/300 column.

Ubiquitin-conjugating enzyme E2 2 (RAD6)
Mol. type   Protein
Organism   Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Olig. state   Monomer
Mon. MW   19.7 kDa
 
UniProt   P06104 (1-172)
Sequence   FASTA
 
PDB ID   1AY
 
PDB ID   1AYZ
 
PDB ID   1AYZ